Lactate proton production

Exercise increases the activity of the purine nucleotide cycle, which converts aspartate to fumarate plus ammonia (see Fig. 41.13). The ammonia is used to buffer the proton production and lactate production from glycolysis, and the fumarate is recycled and can form glutamine. 

An important point to note is that this the above reaction produces lactate, not lactic acid. Nonetheless, protons are produced in glycolysis but in another reaction (Appendix 6.5). Consequently, the two end-products are lactate plus protons, which can be described as lactic acid. Despite this discussion, it can be argued that lactate dehydrogenase is not the terminal reaction of glycolysis, since the lactate plus protons have to be transported out of the cell into the interstitial space. This requires a transporter protein, which transports both lactate and protons across the plasma membrane and out of the cell. 

Assays are frequently needed to detect marked and acute cytotoxicity that may confound the interpretation of cell-based efficacy assays. Neutral red uptake is one of the most commonly used cytotoxicity assays and is used in the regulatory phototoxicity assay on NT3 fibroblasts [13]. It has been show to be more sensitive than assays for mitochondrial reductive capacity such as the tetrazolium reductase assays, ATP depletion assays, or for cell permeabilization or mpture such as dye uptake or lactate dehydrogenase leakage. Lysosomes take up, protonate and trap neutral red when cellular ATP production is sufficient to maintain pH gradients. 

In animal fermentations, an organic molecule (e.g., pyruvate) serves as a terminal proton and electron acceptor, forming an organic end product (e.g., lactate). In contrast, 02 is required as a terminal acceptor for the complete oxidation of substrates such as glucose, glycogen, fatty acids, or amino acids. As discussed in chapter 3, 02 was not always available as one of the substrates for oxidative metabolism and organisms in primordial times had to rely on anaerobic metabolic processes. 

The conversion of substrate to product also requires immobilized water molecules within the active site and an appropriate charge environment to facilitate the transfer of electrons and hydrogens to pyruvate. After pyruvate is bound, its reduction to lactate involves addition of two electrons, one proton, and one hydride ion (H ). NADH provides the electrons and hydride ion the proton comes from the imidazole ring of His-193. The rate-determining step in the covalent chemistry taking place in the catalytic vacuole is that of hydride transfer, which occurs at a rate of approximately 750 s 1 at room temperature for bovine A4-LDH (Dunn et al., 1991). 

Figure 9.11. Three reactive trajectories demonstrate unique pathways from reactants to products for the lactate dehydrogenase enzymatic reaction. The distance of the proton from the NE2 reactive atom of the active site histidine versus the distance of the hydride from the NC4 reactive atom of the coenzyme nicotinamide is plotted, (b) Reactive trajectory...

That lactic acid is the end product of anaerobic glycolysis in muscle tissue has been known for all of this century (Fig. 1). Cell-free extracts able to catalyze the oxidation of lactate to pyruvate were first obtained in 1932 (5). Warburg (6) and von Euler (7) and their colleagues discovered the above reaction [Eq. (1)] and associated it with the chemical properties of a coenzyme. Racker (8) demonstrated in 1950 that the forward reaction also involved the release of a proton. The first purified enzyme was reported by Straub (9) in 1940, while the first micrographs of LDH crystals were shown by Kubowitz and Ott (10). 

The assay based on the observation of NADH production from lactate and NAD+ has also been used 101). The NADH can be detected from its ultraviolet spectrum, but may also be coupled with phenazine metho-sulfate and a tetrazolium salt to give a blue color suitable for automated analysis and histological stains 102). The equilibrium is vmfavorable at neutral pH and the reaction is best carried out at pH 9-10. The reaction can also be followed by observing the proton uptake in a pH-stat 103). 

One of the early findings in experimental ischemia was the production of lactate and a concomitant proton which leads to tissue acidification (Lowry et al., 1964). The activation of anaerobic glycolysis produces tissue acidification which led to an... 

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